菜心下胚轴原生质体培养和植株再生

以萌发３—４ 天（长约４ ｃｍ ）的菜心（Ｂｒａｓｓｉｃａ ｃａｍｐｅｓｔｒｉｓ ｖａｒ．ｐａｒａｃｈｉｎｅｓｉｓ）无菌苗苍白下胚轴为材料,酶解分离原生质体。经纯化的原生质体,在含０．５ ｍ ｇ／ＬＺＴ、０．５ ｍ ｇ／Ｌ２,４－Ｄ、１．０ ｍ ｇ／ＬＮＡＡ 和０．４ ｍ ｏｌ／Ｌ葡萄糖的Ｋ８ｐ 培养基中,进行微滴培养。在起始培养１４—１８小时,原生质体再生新的细胞壁。３６ 小时再生细胞开始第一次分裂。第三天分裂细胞频率可达３５％ 。培养第８—９ 天,可见含８—１６个细胞的小细胞团,植板率为１５％ —１８％ 。３ 周后将发育成直径为２ ｍ ｍ 的白色小愈伤组织,转到含０．３ ｍ ｇ／Ｌ ２,４－Ｄ并用ｇｅｌｒｉｔｅ半固化的培养基上,增殖成４—５ ｍ ｍ 直径的愈伤组织。在ＭＳ＋ ３．２（或１．６） ｍ ｇ／Ｌ ＢＡ＋ １．６（或０．８） ｍ ｇ／ＬＺＴ＋ ０．０１ ｍ ｇ／Ｌ ＮＡＡ＋ ０．１ ｍ ｇ／ＬＧＡ３ 和０．２％ 蔗糖的分化培养基上,获得芽的分化。切下约２ ｃｍ 长的芽苗,转移到含０．２ ｍ ｇ／ＬＩＡＡ 和２％ 蔗糖的培养基上,生根形成完整植株     

马尾松成熟合子胚的体细胞胚胎发生和植株再生

采用马尾松（Ｐｉｎｕｓｍ ａｓｓｏｎｉａｎａ Ｌａｍ ｂ．）成熟合子胚为起始外植体,在含２,４－Ｄ １０ ｍ ｇ／Ｌ,ＫＴ和ＢＡ 各４ ｍ ｇ／Ｌ的ＤＣＲ培养基上得到胚发生培养物。将白色半透明的愈伤组织（含早期原胚）在含２,４－Ｄ１．０ ｍ ｇ／Ｌ,ＫＴ和ＢＡ 各０．４ ｍ ｇ／Ｌ的ＤＣＲ培养基上保持并增殖。在附加９０００ ｍ ｇ／Ｌ肌醇的ＤＣＲ高渗培养基上得到粗壮的后期原胚。ＡＢＡ 和活性炭同时使用能促进子叶胚的形成,最高频率为３５．１％ 。在无激素培养基上,成熟体细胞胚萌发并进一步形成完整小植株     

提高菜心离体植株再生频率的研究

ＡｇＮＯ３与ＡＢＡ 相配合,在离体培养条件下从３个菜心（Ｂｒａｓｓｉｃａ ｐａｒａｃｈｉｎｅｎｓｉｓＢａｉｌ．）品种（“４９－１９”、“６０Ｄ”和“７０Ｄ”）获得了高频率的植株再生。结果表明：在试验的３种外植体中,子叶－子叶柄再生能力最强。实生苗龄及接种方式影响植株再生频率。ＭＳ附加不同浓度的ＢＡＰ和ＮＡＡ,ＢＡＰ ２ ｍ ｇ／Ｌ与ＮＡＡ １ ｍ ｇ／Ｌ配合效果最好,植株再生频率可达２６．８％ 。培养基中单独添加ＡｇＮＯ３ 或ＡＢＡ 均能促进植株再生,且ＡｇＮＯ３ 作用优于ＡＢＡ。ＡｇＮＯ３（４ ｍ ｇ／Ｌ）和ＡＢＡ（０．５ ｍ ｇ／Ｌ）相配合获得了更高的植株再生频率：“４９－１９”达８９％ ,“６０Ｄ”达８４．３％ ,“７０Ｄ”达８６％ 。组织学研究表明,在本实验条件下菜心离体培养植株再生方式为外植体直接出芽。不定芽起源于子叶柄切口端维管组织的薄壁细胞;由此种细胞分裂形成分生细胞团,继而分化成芽。试管苗移入盆中获得成熟植株     

苹果原生质体培养及植株再生

用苹果（Ｍａｌｕｓ ｐｕｍ ｉｌａ Ｍｉｌｌ）胚珠诱导愈伤组织并建立悬浮细胞系。用２％ 纤维素酶、０．５％ 果胶酶的混合酶液分离悬浮培养物,可得到５．４×１０６ 个／ｇ ｆｒ． ｗ ｔ有活性的原生质体。这些原生质体在改良的ＭＳ、Ｋ８ｐ、Ｄ２ 培养基中均可发育成细胞团,在含２．０ ｍ ｇ／ＬＩＡＡ、２．０ｍ ｇ／ＬＮＡＡ、０．１ ｍ ｇ／ＬＢＡ 的ＭＳ固体培养基上形成愈伤组织,更换几次不同的培养基后,在分化培养基上分化出不定芽,在生根培养基上生根形成完整植株。     

石斛离体培养中ABA对诱导花芽形成的影响

由兰科植物铁皮石斛（Ｄｅｎｄｒｏｂｉｕｍ ｃａｎｄｉｄｕｍ Ｗａｌｌ．ｅｘ Ｌｉｎｄｌ．）种子诱导形成的愈伤组织,在光照下置于ＭＳ附加０．３ ｍ ｇ／ＬＮＡＡ 的培养基上繁殖,可以形成原球茎。将原球茎转入ＭＳ含２ ｍ ｇ／Ｌ ６－ＢＡ 和０．５ ｍ ｇ／ＬＮＡＡ 的培养基上,花芽形成频率为２７．０％ 。原球茎先在０．５ ｍ ｇ／ＬＡＢＡ的培养基上预培养１５ ｄ,再转入含２ ｍ ｇ／Ｌ６－ＢＡ 的ＭＳ培养基上培养,花芽形成频率明显提高,可达８４．４％ ,而且每株植株花的数目增加;但是在仅有ＡＢＡ 的ＭＳ培养基上培养的原球茎再生的植株未见花芽形成     

杨树新品种叶肉原生质体培养和植株再生

从１ 个月龄的ＮＬ－８０１０６ 杨（Ｐｏｐｕｌｕｓｄｅｌｔｏｉｄｅｓ×Ｐ． ｓｉｍ ｏｎｉｉ）无菌苗叶片分离得到大量原生质体,纯化后其原生质体产量为４×１０７／ｇ ｆｒ．ｗ ｔ． 纯化的原生质体在含２,４－Ｄ ２ ｍ ｇ／Ｌ、ＮＡＡ ０．５ ｍ ｇ／Ｌ和ＫＴ ０．５ ｍ ｇ／Ｌ的ＫＭ８ｐ 和ＭＳ培养基中进行高密度液体浅层培养,渗透势为０．４０ ｍ ｏｌ／Ｌ的ＫＭ８ｐ 培养基中原生质体分裂频率最高． 培养第５ 天观察到第一次细胞分裂,培养１０ ｄ 的分裂频率为４．５％ ,１２ 周内可形成大量的细胞团和小愈伤组织． ＮＬ－８０１０６杨叶肉原生质体在富含有机氮并以葡萄糖为碳源的培养基中具有较高的分裂频率和植板率．小愈伤组织在ｇｅｌｒｉｔｅ 固化的ＮＬＺ１ 培养基上增殖生长,３ 周后形成４—６ ｍ ｍ 结构紧密的鲜红色愈伤组织,转至ＮＬＦ分化培养基,分化成苗率为１００％ ． 待芽伸长到３ ｃｍ 时,从基部切下转至１／２ ＭＳ培养基上诱导生根,形成完整植株     

亚麻下胚轴离体培养和转化的研究

报道了亚麻（Ｌｉｎｕｍ　ｕｓｉｔａｔｉｓｓｉｍｕｍ）的高频率植株再生和细胞转化。亚麻下胚轴切段培养在ＭＳＢ分化培养基上,诱导分化出不定芽。最佳的激素组合是ＢＡ２ｍｇ／Ｌ＋ＩＡＡ０．５ｍｇ／Ｌ,分化频率可达９７％。在附加ＢＡ１ｍｇ／Ｌ和ＮＡＡ０．０２ｍｇ／Ｌ的ＭＳＢ培养基上,亚麻下胚轴外植体的不定芽分化频率也可达９０％以上。用根癌农杆菌（Ａｇｒｏｈａｃｔｅｒｉｕｍ　ｔｕｍｅｆａｃｉｅｎｓ）感染亚麻下胚轴外植体,在含卡那霉素５０ｍｇ／Ｌ的选择培养基上筛选获得卡那霉素抗性苗,并检测到ＧＵＳ基因活性表达。表明它们是转化植株,转化频率约为２％。     

硬粒小麦单倍体原生质体培养及植株再生

由硬粒小麦（Ｔｒｉｔｉｃｕｍ ｄｕｒｕｍ Ｄｅｓｆ．）×玉米（Ｚｅａ ｍａｙｓＬ．）建立的单倍性胚性愈伤组织,在继代培养４ 个月后置于含２．０ ｍ ｇ／Ｌ２,４－Ｄ、３％ 蔗糖、２００ ｍ ｇ／Ｌ水解酪蛋白、１４６ ｍ ｇ／Ｌ谷氨酰胺和３００ ｍ ｇ／Ｌ天冬氨酸的ＭＳ液体培养基中进行悬浮培养,４ 个月后形成了生长迅速、由大小不同（０．５ ～５ ｍ ｍ ）的愈伤组织块组成的愈伤组织悬浮系。酶解试验表明,２．０％ 纤维素酶ＲＳ和０．５％ 的离析酶效果最好,而液体悬浮培养物和固体培养的愈伤组织（在酶解时用锋利的解剖刀片切成１ ｍ ｍ 左右的小块）都能释放出大量原生质体,但悬浮培养物释放出的原生质体状态较好,胞质更浓厚,用ＫＭ８ｐ 培养基以琼脂糖包埋培养方式培养时分裂频率可达５％ 左右。由原生质体再生的小愈伤组织经增殖、筛选后可获得胚性愈伤组织,将其转移至分化培养基Ⅰ（０．２ ｍ ｇ／Ｌ ２,４－Ｄ、１．０ ｍ ｇ／Ｌ ＢＡＰ、０．１ ｍ ｇ／ＬＮＡＡ、３％ 蔗糖、２００ ｍ ｇ／Ｌ 水解酪蛋白、１４６ ｍ ｇ／Ｌ谷氨酰胺和３００ ｍ ｇ／Ｌ天冬氨酸的ＭＳ固体培养基）和Ⅱ（不含２,４－Ｄ,其它成分同Ⅰ）上进行分步分化培养可再生出完整植株,分化频率约为２０％     

根癌农杆菌对甘蓝型油菜的转化及转基因植株的再生

用根癌农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍ ｅｆａｃｉｅｎｓ）共培养法把外源基因导入甘蓝型油菜（Ｂｒａｓｓｉ－ｃａ ｎａｐｕｓＬ．）主要栽培品种“云北２ 号”,获得转基因植株。所用外植体为带有１—２ ｍ ｍ 子叶柄的完整子叶,根癌农杆菌为Ａ２０８ＳＥ（ｐＴｉＴ３７－ＳＥ, ｐＲＯＡ９３）。Ｔｉ质粒ｐＲＯＡ９３ 带有ＮＰＴⅡ及ＧＵＳ嵌合基因。共培养２ 天后转到附加２５ ｍ ｇ／Ｌ卡那霉素的分化培养基（ＭＳ＋ ４．５ ｍ ｇ／ＬＢＡＰ）上。ＡｇＮＯ３ 和羧苄青霉素促进芽的分化,头孢霉素则有抑制作用。最高转化频率为２７％ 。把分化出的茎芽切下,插入含有２５ ｍ ｇ／Ｌ卡那霉素的生根培养基中。羧苄青霉素不利于根的形成。把完整抗性植株移入盛土壤的盆中,生长状况良好。测定β－葡糖苷酸酶活性,８４％ 明显高于对照。以ＮＰＴⅡ基因作探针进行Ｓｏｕｔｈｅｒｎ ｂｌｏｔ分析,证实外源基因已插入到植物细胞基因组中     

苎麻原生质体培养及植株再生

用苎麻（Ｂｏｅｈｍ ｅｒｉａ ｎｉｖｅａ）子叶诱导愈伤组织并建立悬浮细胞系． 用４．５％ 纤维素酶、０．８％ 离析酶、０．８％ 半纤维素酶的混合酶液分离悬浮培养细胞,可得到２×１０６ 个／ｇ ｆｒ．ｗｔ的原生质体．这些原生质体以海藻酸钠包埋方式培养在附加２,４－Ｄ ０．５ ｍ ｇ／Ｌ、ＫＴ ０．５ ｍ ｇ／Ｌ 的ＫＭ８ｐ 培养基中,５０ ｄ 左右可形成肉眼可见的小愈伤组织．愈伤组织经过扩增,在不同的分化培养基上可诱导芽、根的形成,再生出完整植株． 子叶原生质体则仅能进行几次有限的分裂     

Promotion of Carrot Suspension Cell Transformation and Plant Regeneration byAgrobacterium Harboring Binary Vector Pretreated with Phenolic Compounds

Non-tumorigenic Agrobacterium tumefaciens strain LBA4404 harboring binary vector pretreated with acetosyringone and complex phenolic compounds significantly increased the rate of carrot (Daucus carota) suspension cell transformation and plant regeneration. Kanamycin resistant colones were selected on media containing 50 mg/L kanamycin. The author's experimental data indicated that transformation frequencies in the group activated by acetosyringone and complex phenolic compounds pretreatment were 0.24% and 0. 17% respectively while that in the phenolic compound non-pretreated was only 0.02%. Transgenic carrot plants were obtained from the resistant colones via somatic embryogenesis on hormone-free media containing 50 mg/L kanamycin. Histochemical analysis showed that high levels of GUS activity appeared in the parenchymatous cells of vascular bundles of roots, stems, leaves and petioles as well as stomatic guard cells, cortical cells, mesophyllous cells and trichome of leaves, also epidermal cells also parenchymatous cells under the epiderm of petiols.     

Factors influencing the Agrobacterium tumefaciens-mediated transformation of carrot (Daucus carota L.)

To develop an efficient procedure for Agrobacterium tumefaciens-mediated genetic transformation of carrot (Daucus carota L.) the effects of several factors were studied. Parameters which significantly affected the transformation frequency were the variety, the explant type, and the co-cultivation period. Under optimal conditions, using the A. tumefaciens C58C1 containing either pGSTRN943 or pGSGluc1 and 3 days of co-cultivation, the frequency of transformation of petiole explants of the variety Nanco was greater than 45%. This procedure does not require acetosyringone or prolonged precultivation period. Using kanamycin (100 mg l^-1) for selection, a large number of transgenic plantlets developed from the embryogenic calli within 8–10 weeks of culture on hormone-free medium. Transformation was confirmed by histochemical detection of -glucuronidase activity in the transformed cells, by the ability of petiole segments to produce embryogenic calli in presence of kanamycin, and by Southern hybridization analyses.     

带内含子卡那霉素抗性基因双元载体构建及烟草转化

农杆菌介导法是植物基因转化的常用方法,然而由于筛选培养基中常用的抗生素头孢霉素和羧苄青霉素具有类植物激素活性,影响外植体的再生和转化频率。将一个植物的内含子插入卡那霉素抗性基因编码区的N端,合成了一个带内含子的卡那霉素抗性基因。构建带该基因的植物双元表达栽体pYP1202并转化烟草,受侵外植体在含卡那霉素50～200mg/L的选择培养基中抗性芽分化频率不受卡那霉素浓度影响,然而具有GUS活性的转化子占分化芽的比例却随着卡那霉素浓度的增加而升高。当培养基中加入500mg/L羧苄青霉素后受侵外植体产生的抗性芽频率比单一的卡那霉素筛选提高近1倍,高达91.4％,然而具GUS活性的转化子占抗性芽的比例仅有26.7％,在200m/L的卡那霉素筛选下,比例升至93.3％。用带内含子卡那霉素抗性基因构建的植物表达载体转化植物可以减少假抗性芽的产生。     

Transformation and regeneration of carrot (Daucus carota L.)

A protocol is presented for the efficient transformation of carrot (Daucus carota L. cv. Nantaise) by Agrobacterium tumefaciens. The binary vector contained the marker gene -glucuronidase (GUS), driven by the 35S promoter of cauliflower mosaic virus, and the nptII gene, which confers kanamycin resistance. Highest T-DNA transfer rates were obtained by co-cultivating bacteria with hypocotyl segments of dark-grown seedlings on solidified B5 medium containing naphthaleneacetic acid and 6-benzylaminopurine. After 2 days, bacterial growth was stopped with antibiotics. Two weeks later, the explants were placed on agar containing the kanamycin derivate geneticin; antibiotic-resistant calli developed during the following 4 weeks. Suspension cultures were obtained from resistant calli and plants regenerated via somatic embryogenesis in liquid culture. The majority of plants were phenotypically normal and, depending on the Agrobacterium strain used, harbored single or multiple copies of the T-DNA. About equal levels of GUS activity were found in different organs of young plants up to 6 weeks after embryogenesis. In leaves of older plants, GUS activity was markedly reduced, whereas the activities in phloem and xylem parenchyma cells of developing tap roots were still high and fairly uniform. Thus, the 35S promoter may be a useful tool to drive the expression of transgenes in developing carrot storage roots.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Dioscorea zingiberensis</Emphasis> Wright,an important pharmaceutical crop

Dioscorea zingiberensis Wright has been cultivated as a pharmaceutical crop for production of diosgenin, a precursor for synthesis of various important steroid drugs. Because breeding of D. zingiberensis through sexual hybridization is difficult due to its unstable sexuality and differences in timing of flowering in male and female plants, gene transfer approaches may play a vital role in its genetic improvement. In this study, the Agrobacterium tumefaciens-mediated transformation of D. zingiberensis was investigated with leaves and calli as explants. The results showed that both leaf segments and callus pieces were sensitive to 30 mg/l hygromycin and 50–60 mg/l kanamycin, and using calli as explants and addition of acetosyringone (AS) in cocultivation medium were crucial for successful transformation. We first immersed callus explants in A. tumefaciens cells for 30 min and then transferred the explants onto a co-cultivation medium supplemented with 200 μM AS for 3 days. Three days after, we cultured the infected explants on a selective medium containing 50 mg/l kanamycin and 100 mg/l timentin for formation of kanamycin-resistant calli. After the kanamycin-resistant calli were produced, we transferred them onto fresh selective medium for shoot induction. Finally, the kanamycin resistant shoots were rooted and the stable incorporation of the transgene into the genome of D. zingiberensis plants was confirmed by GUS histochemical assay, PCR and Southern blot analyses. The method reported here can be used to produce transgenic D. zingiberensis plants in 5 months and the transformation frequency is 24.8% based on the numbers of independent transgenic plants regenerated from initial infected callus explants.     

Tomato Transformation and Transgenic Plant Production

Tomato transformation and regeneration were analysed and optimized. Cotyledon explants from Lycopersicon esculentum cv. UC82B, were infected by Agrobacterium tumefaciens strain LBA4404 harbouring the neomycin phosphotransferase (NPTII) reporter gene. The effects of phenolic compounds, vitamins and growth regulators on plant transformation and regeneration were studied. Increasing the vitamin thiamine concentration from 0.1 mg l^–1 in standard medium to 0.4 mg l^–1 decreased the chlorophyll lost that accompanied the expansion of necrotic areas in cotyledon explants. Optimal shoot regeneration rate was obtained with a balanced concentration of 0.5 mg l^–1 auxin indolelacetic acid (IAA) and 0.5 mg l^–1 cytokinin zeatin riboside. Finally, when the phenolic acetosyringone was present in the co-culture medium at 200 µM, confirmed transgenic lines reached 50% of antibiotic resistant shoots. Under the above conditions, the transformation efficiency reached 12.5%.     

Agrobacterium tumefaciens-mediated transformation of rutabaga (Brassica napus var. napobrassica) cultivar “American Purple Top Yellow”

An efficient protocol for genetic transformation of rutabaga (Brassica napus var. napobrassica) cultivar ??American Purple Top Yellow?? was developed by optimizing several factors influencing gene delivery and plant regeneration. A two-step regeneration protocol, adapted from canola, was optimal for rutabaga regeneration using hypocotyl explants. Transient expression studies monitored by histochemical ??-glucuronidase (GUS) assays indicated that several factors, including Agrobacterium tumefaciens strain, cocultivation time, and cocultivation medium, affected gene delivery. For stable transformation, precultured hypocotyl explants were cocultivated with Agrobacterium cells on sterilized filter paper overlaid on callus induction medium containing 100???M acetosyringone for 6?d under a 16-h photoperiod. Selection and regeneration of transformed cells were conducted on media containing 50?mg?l^?1 kanamycin and 250?mg?l^?1 Timentin. Using this protocol, GUS- and PCR-positive transformants were obtained from 3.2 to 4.2?% of hypocotyl explants inoculated with each of the three Agrobacterium strains after 3?C5?mo. Most transformants exhibited a normal phenotype. Southern blot analysis confirmed stable integration of the gusA transgene in T₀ plants.     

根癌农杆菌介导蓝猪耳转化的影响因素研究

研究了根癌农杆菌介导蓝猪耳转化的影响因子。结果表明,以蓝色花类型蓝猪耳5~6周的叶片为外植体,在OD₆₀₀为0.5~0.6的活化菌液中浸染5~10min,暗培养4d后,在愈伤组织诱导培养基(MS+BA 1.0mg/L+2,4-D 0.1mg/L)上生长14d,获得抗性愈伤组织;经芽诱导培养基(1/2MS+BA 1.0mg/L+NAA 0.1mg/L)培养28d得到抗性芽;生根培养基(1/2MS)上培养14d得到抗性植株。经PCR检测证实外源基因已整合到蓝猪耳基因组中,转化率达13%~14%。Cef和Hyg浓度对转化影响较大,转化的不同阶段其适宜浓度不同。     

发根农杆菌LBA9402Bin19转化红豆草及再生转基因植株

Hypocotyl segments of Onobrychis viciaefolia were transformed by Agrobacterium rhizogenes LBA9402 which harboured pBin19 and pRi1855. Seedling age and preculture time of hypocotyl segments influenced the transformation frequency. Paper electrophoresis revealed that 70% of single hairy root cultures could synthesize agropine. Calli were induced from hairy root segments on MS medium containing 0-9.05 mumol/L 2,4-D and 0-2.22 mumol/L 6-BA at first, then they were transferred onto MS0 medium without kanamycin for regeneration. Constitution and concentration of phytohormones in callus induction media affected subsequent regeneration of calluses on MS0 medium remarkably. Regeneration frequency and shoot number per callus declined when 2,4-D concentration in callus induction media increased from 4.52 to 9.05 mumol/L, while they ascended when 6-BA in callus induction media increased from 0 to 2.22 mumol/L. On MS medium supplemented with 4.52 mumol/L 2,4-D and 2.22 mumol/L 6-BA, only 14.2% hairy root segments could produce calluses, but the regeneration frequency reached 58.1% and the shoot number per callus was 37.2. In 32 analysed plants regenerated from 8 kanamycin resistant hairy root lines, 25 were nptII positive and showed different copy numbers.